Lipid-mediated polynucleotide administration to deliver a biologically active peptide and to induce a cellular immune response

ABSTRACT

A method for delivering an isolated polynucleotide to the interior of a cell in a vertebrate, comprising the interstitial introduction of an isolated polynucleotide into a tissue of the vertebrate where the polynucleotide is taken up by the cells of the tissue and exerts a therapeutic effect on the vertebrate. The method can be used to deliver a therapeutic polypeptide to the cells of the vertebrate, to provide an immune response upon in vivo translation of the polynucleotide, to deliver antisense polynucleotides, to deliver receptors to the cells of the vertebrate, or to provide transitory gene therapy.

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 467,881 filed Jan. 19, 1990, which is a continuation-in-part ofU.S. application Ser. No. 326,305 filed Mar. 21, 1989.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to introduction of naked DNA andRNA sequences into a vertebrate to achieve controlled expression of apolypeptide. It is useful in gene therapy, vaccination, and anytherapeutic situation in which a polypeptide should be administered tocells in vivo.

[0003] Current research in gene therapy has focused on “permanent”cures, in which DNA is integrated into the genome of the patient. Viralvectors are presently the most frequently used means for transformingthe patient's cells and introducing DNA into the genome. In an indirectmethod, viral vectors, carrying new genetic information, are used toinfect target cells removed from the body, and these cells are thenre-implanted. Direct in vivo gene transfer into postnatal animals hasbeen reported for formulations of DNA encapsulated in liposomes and DNAentrapped in proteoliposomes containing viral envelope receptor proteins(Nicolau et al., Proc. Natl. Acad Sci USA 80:1068-1072 (1983); Kaneda etal., Science 243:375-378 (1989); Mannino et al., Biotechniques 6:682-690(1988). Positive results have also been described with calcium phosphateco-precipitated DNA (Benvenisty and Reshef Proc. Natl. Acad Sci USA83:9551-9555 (1986)).

[0004] The clinical application of gene therapy, as well as theutilization of recombinant retrovirus vectors, has been delayed becauseof safety considerations. Integration of exogenous DNA into the genomeof a cell can cause DNA damage and possible genetic changes in therecipient cell that could predispose to malignancy. A method whichavoids these potential problems would be of significant benefit inmaking gene therapy safe and effective.

[0005] Vaccination with immunogenic proteins has eliminated or reducedthe incidence of many diseases; however there are major difficulties inusing proteins associated with other pathogens and disease states asimmunogens. Many protein antigens are not intrinsically immunogenic.More often, they are not effective as vaccines because of the manner inwhich the immune system operates.

[0006] The immune system of vertebrates consists of several interactingcomponents. The best characterized and most important parts are thehumoral and cellular (cytolytic) branches. Humoral immunity involvesantibodies, proteins which are secreted into the body fluids and whichdirectly recognize an antigen. The cellular system, in contrast, relieson special cells which recognize and kill other cells which areproducing foreign antigens. This basic functional division reflects twodifferent strategies of immune defense. Humoral immunity is mainlydirected at antigens which are exogenous to the animal whereas thecellular system responds to antigens which are actively synthesizedwithin the animal.

[0007] Antibody molecules, the effectors of humoral immunity, aresecreted by special B lymphoid cells, B cells, in response to antigen.Antibodies can bind to and inactivate antigen directly (neutralizingantibodies) or activate other cells of the immune system to destroy theantigen.

[0008] Cellular immune recognition is mediated by a special class oflymphoid cells, the cytotoxic T cells. These cells do not recognizewhole antigens but instead they respond to degraded peptide fragmentsthereof which appear on the surface of the target cell bound to proteinscalled class I major histocompatibility complex (MHC) molecules.Essentially all nucleated cells have class I molecules. It is believedthat proteins produced within the cell are continually degraded topeptides as part of normal cellular metabolism. These fragments arebound to the MHC molecules and are transported to the cell surface. Thusthe cellular immune system is constantly monitoring the spectra ofproteins produced in all cells in the body and is poised to eliminateany cells producing foreign antigens.

[0009] Vaccination is the process of preparing an animal to respond toan antigen. Vaccination is more complex than immune recognition andinvolves not only B cells and cytotoxic T cells but other types oflymphoid cells as well. During vaccination, cells which recognize theantigen (B cells or cytotoxic T cells) are clonally expanded. Inaddition, the population of ancillary cells (helper T cells) specificfor the antigen also increase. Vaccination also involves specializedantigen presenting cells which can process the antigen and display it ina form which can stimulate one of the two pathways.

[0010] Vaccination has changed little since the time of Louis Pasteur. Aforeign antigen is introduced into an animal where it activates specificB cells by binding to surface immunoglobulins. It is also taken up byantigen processing cells, wherein it is degraded, and appears infragments on the surface of these cells bound to Class II MHC molecules.Peptides bound to class II molecules are capable of stimulating thehelper class of T cells. Both helper T cells and activated B cells arerequired to produce active humoral immunization. Cellular immunity isthought to be stimulated by a similar but poorly understood mechanism.

[0011] Thus two different and distinct pathways of antigen processingproduce exogenous antigens bound to class II MHC molecules where theycan stimulate T helper cells, as well as endogenous proteins degradedand bound to class I MHC molecules and recognized by the cytotoxic classof T cells.

[0012] There is little or no difference in the distribution of MHCmolecules. Essentially all nucleated cells express class I moleculeswhereas class II MHC proteins are restricted to some few types oflymphoid cells.

[0013] Normal vaccination schemes will always produce a humoral immuneresponse. They may also provide cytotoxic immunity. The humoral systemprotects a vaccinated individual from subsequent challenge from apathogen and can prevent the spread of an intracellular infection if thepathogen goes through an extracellular phase during its life cycle;however, it can do relatively little to eliminate intracellularpathogens. Cytotoxic immunity complements the humoral system byeliminating the infected cells. Thus effective vaccination shouldactivate both types of immunity.

[0014] A cytotoxic T cell response is necessary to remove intracellularpathogens such as viruses as well as malignant cells. It has provendifficult to present an exogenously administered antigen in adequateconcentrations in conjunction with Class I molecules to assure anadequate response. This has severely hindered the development ofvaccines against tumor-specific antigens (e.g., on breast or coloncancer cells), and against weakly immunogenic viral proteins (e.g., HIV,Herpes, non-A, non-B hepatitis, CMV and EBV).

[0015] It would be desirable to provide a cellular immune response alonein immunizing against agents such as viruses for which antibodies havebeen shown to enhance infectivity. It would also be useful to providesuch a response against both chronic and latent viral infections andagainst malignant cells.

[0016] The use of synthetic peptide vaccines does not solve theseproblems because either the peptides do not readily associate withhistocompatibility molecules, have a short serum half-life, are rapidlyproteolyzed, or do not specifically localize to antigen-presentingmonocytes and macrophages. At best, all exogenously administeredantigens must compete with the universe of self-proteins for binding toantigen-presenting macrophages.

[0017] Major efforts have been mounted to elicit immune responses topoorly immunogenic viral proteins from the herpes viruses, non-A, non-Bhepatitis, HIV, and the like. These pathogens are difficult andhazardous to propagate in vitro. As mentioned above, synthetic peptidevaccines corresponding to viral-encoded proteins have been made, buthave severe pitfalls. Attempts have also been made to use vaccinia virusvectors to express proteins from other viruses. However, the resultshave been disappointing, since (a) recombinant vaccinia viruses may berapidly eliminated from the circulation in already immune individuals,and (b) the administration of complex viral antigens may induce aphenomenon known as “antigenic competition,” in which weakly immunogenicportions of the virus fail to elicit an immune response because they areout-competed by other more potent regions of the administered antigen.

[0018] Another major problem with protein or peptide vaccines isanaphylactic reaction which can occur when injections of antigen arerepeated in efforts to produce a potent immune response. In thisphenomenon, IgE antibodies formed in response to the antigen causesevere and sometimes fatal allergic reactions.

[0019] Accordingly, there is a need for a method for invoking a safe andeffective immune response to this type of protein or polypeptide.Moreover, there is a great need for a method that will associate theseantigens with Class I histocompatibility antigens on the cell surface toelicit a cytotoxic T cell response, avoid anaphylaxis and proteolysis ofthe material in the serum, and facilitate localization of the materialto monocytes and macrophages.

[0020] A large number of disease states can benefit from theadministration of therapeutic peptides. Such peptides includelymphokines, such as interleukin-2, tumor necrosis factor, and theinterferons; growth factors, such as nerve growth factor, epidermalgrowth factor, and human growth hormone; tissue plasminogen activator;factor VIII:C; granulocyte-macrophage colony-stimulating factor;erythropoietin; insulin; calcitonin; thymidine kinase; and the like.Moreover, selective delivery of toxic peptides (such as ricin,diphtheria toxin, or cobra venom factor) to diseased or neoplastic cellscan have major therapeutic benefits. Current peptide delivery systemssuffer from significant problems, including the inability to effectivelyincorporate functional cell surface receptors onto cell membranes, andthe necessity of systemically administering large quantities of thepeptide (with resultant undesirable systemic side effects) in order todeliver a therapeutic amount of the peptide into or onto the targetcell.

[0021] These above-described problems associated with gene therapy,immunization, and delivery of therapeutic peptides to cells areaddressed by the present invention.

DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 comprises autoradiograms of chromatographic studies showingthe expression of the CAT gene in mouse muscle.

[0023]FIG. 2 comprises photomicrographs of muscle tissue stained forbeta-galactosidase activity following injection with the pRSVLac-Z DNAvector.

[0024]FIG. 3 presents data for luciferase activity in muscle followingthe injection of βgLucβgA_(n) into muscle.

[0025]FIG. 4 presents an autoradiogram of a Southern blot after analysisof extracts from pRSVL-injected muscle.

[0026]FIG. 5 comprises graphs showing antibody production in micefollowing the injection of a gene for an immunogenic peptide.

[0027]FIG. 6 comprises graphs showing antibody production in micefollowing the injection of mouse cells transfected with a gene for animmunogenic peptide.

SUMMARY OF THE INVENTION

[0028] The present invention provides a method for delivering apharmaceutical or immunogenic polypeptide to the interior of a cell of avertebrate in vivo, comprising the step of introducing a preparationcomprising a pharmaceutically acceptable injectable carrier and a nakedpolynucleotide operatively coding for the polypeptide into theinterstitial space of a tissue comprising the cell, whereby the nakedpolynucleotide is taken up into the interior of the cell and has animmunogenic or pharmacological effect on the vertebrate. Also providedis a method for introducing a polynucleotide into muscle cells in vivo,comprising the steps of providing a composition comprising a nakedpolynucleotide in a pharmaceutically acceptable carrier, and contactingthe composition with muscle tissue of a vertebrate in vivo, whereby thepolynucleotide is introduced into muscle cells of the tissue. Thepolynucleotide may be an antisense polynucleotide. Alternatively, thepolynucleotide may code for a therapeutic peptide that is expressed bythe muscle cells after the contacting step to provide therapy to thevertebrate. Similarly, it may code for an immunogenic peptide that isexpressed by the muscle cells after the contacting step and whichgenerates an immune response, thereby immunizing the vertebrate.

[0029] One particularly attractive aspect of the invention is a methodfor obtaining long term administration of a polypeptide to a vertebrate,comprising the step of introducing a naked DNA sequence operativelycoding for the polypeptide interstitially into tissue of the vertebrate,whereby cells of the tissue produce the polypeptide for at least onemonth or at least 3 months, more preferably at least 6 months. In thisembodiment of the invention, the cells producing the polypeptide arenonproliferating cells, such as muscle cells.

[0030] Another method according to the invention is a method forobtaining transitory expression of a polypeptide in a vertebrate,comprising the step of introducing a naked mRNA sequence operativelycoding for the polypeptide interstitially into tissue of the vertebrate,whereby cells of the tissue produce the polypeptide for less than about20 days, usually less than about 10 days, and often less than 3 or 5days. For many of the methods of the invention, administration intosolid tissue is preferred.

[0031] One important aspect of the invention is a method for treatmentof muscular dystrophy, comprising the steps of introducing a therapeuticamount of a composition comprising a polynucleotide operatively codingfor dystrophin in a pharmaceutically acceptable injectable carrier invivo into muscle tissue of an animal suffering from muscular dystrophy,whereby the polynucleotide is taken up into the cells and dystrophin isproduced in vivo. Preferably, the polynucleotide is a nakedpolynucleotide and the composition is introduced interstitially into themuscle tissue.

[0032] The present invention also includes pharmaceutical products forall of the uses contemplated in the methods described herein. Forexample, there is a pharmaceutical product, comprising nakedpolynucleotide, operatively coding for a biologically activepolypeptide, in physiologically acceptable administrable form, in acontainer, and a notice associated with the container in form prescribedby a governmental agency regulating the manufacture, use, or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the polynucleotide for human or veterinary administration.Such notice, for example, may be the labeling approved by the U.S. Foodand Drug Administration for prescription drugs, or the approved productinsert.

[0033] In another embodiment, the invention provides a pharmaceuticalproduct, comprising naked polynucleotide, operatively coding for abiologically active peptide, in solution in a physiologically acceptableinjectable carrier and suitable for introduction interstitially into atissue to cause cells of the tissue to express the polypeptide, acontainer enclosing the solution, and a notice associated with thecontainer in form prescribed by a governmental agency regulating themanufacture, use, or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of manufacture, use, or sale of the solutionof polynucleotide for human or veterinary administration. The peptidemay be immunogenic and administration of the solution to a human mayserve to vaccinate the human, or an animal. Similarly, the peptide maybe therapeutic and administration of the solution to a vertebrate inneed of therapy relating to the polypeptide will have a therapeuticeffect.

[0034] Also provided by the present invention is a pharmaceuticalproduct, comprising naked antisense polynucleotide, in solution in aphysiologically acceptable injectable carrier and suitable forintroduction interstitially into a tissue to cause cells of the tissueto take up the polynucleotide and provide a therapeutic effect, acontainer enclosing the solution, and a notice associated with thecontainer in form prescribed by a governmental agency regulating themanufacture, use, or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of manufacture, use, or sale of the solutionof polynucleotide for human or veterinary administration.

[0035] One particularly important aspect of the invention relates to apharmaceutical product for treatment of muscular dystrophy, comprising asterile, pharmaceutically acceptable carrier, a pharmaceuticallyeffective amount of a naked polynucleotide operatively coding fordystrophin in the carrier, and a container enclosing the carrier and thepolynucleotide in sterile fashion. Preferably, the polynucleotide isDNA.

[0036] From yet another perspective, the invention includes apharmaceutical product for use in supplying a biologically activepolypeptide to a vertebrate, comprising a pharmaceutically effectiveamount of a naked polynucleotide operatively coding for the polypeptide,a container enclosing the carrier and the polynucleotide in a sterilefashion, and means associated with the container for permitting transferof the polynucleotide from the container to the interstitial space of atissue, whereby cells of the tissue can take up and express thepolynucleotide. The means for permitting such transfer can include aconventional septum that can be penetrated, e.g., by a needle.Alternatively, when the container is a syringe, the means may beconsidered to comprise the plunger of the syringe or a needle attachedto the syringe. Containers used in the present invention will usuallyhave at least 1, preferably at least 5 or 10, and more preferably atleast 50 or 100 micrograms of polynucleotide, to provide one or moreunit dosages. For many applications, the container will have at least500 micrograms or 1 milligram, and often will contain at least 50 or 100milligrams of polynucleotide.

[0037] Another aspect of the invention provides a pharmaceutical productfor use in immunizing a vertebrate, comprising a pharmaceuticallyeffective amount of a naked polynucleotide operatively coding for animmunogenic polypeptide, a sealed container enclosing the polynucleotidein a sterile fashion, and means associated with the container forpermitting transfer of the polynucleotide from the container to theinterstitial space of a tissue, whereby cells of the tissue can take upand express the polynucleotide.

[0038] Still another aspect of the present invention is the use of nakedpolynucleotide operatively coding for a physiologically activepolypeptide in the preparation of a pharmaceutical for introductioninterstitially into tissue to cause cells comprising the tissue toproduce the polypeptide. The pharmaceutical, for example, may be forintroduction into muscle tissue whereby muscle cells produce thepolypeptide. Also contemplated is such use, wherein the peptide isdystrophin and the pharmaceutical is for treatment of musculardystrophy.

[0039] Another use according to the invention is use of naked antisensepolynucleotide in the preparation of a pharmaceutical for introductioninterstitially into tissue of a vertebrate to inhibit translation ofpolynucleotide in cells of the vertebrate.

[0040] The tissue into which the polynucleotide is introduced can be apersistent, non-dividing cell. The polynucleotide may be either a DNA orRNA sequence. When the polynucleotide is DNA, it can also be a DNAsequence which is itself non-replicating, but is inserted into aplasmid, and the plasmid further comprises a replicator. The DNA may bea sequence engineered so as not to integrate into the host cell genome.The polynucleotide sequences may code for a polypeptide which is eithercontained within the cells or secreted therefrom, or may comprise asequence which directs the secretion of the peptide.

[0041] The DNA sequence may also include a promoter sequence. In onepreferred embodiment, the DNA sequence includes a cell-specific promoterthat permits substantial transcription of the DNA only in predeterminedcells. The DNA may also code for a polymerase for transcribing the DNA,and may comprise recognition sites for the polymerase and the injectablepreparation may include an initial quantity of the polymerase.

[0042] In many instances, it is preferred that the polynucleotide istranslated for a limited period of time so that the polypeptide deliveryis transitory. The polypeptide may advantageously be a therapeuticpolypeptide, and may comprise an enzyme, a hormone, a lymphokine, areceptor, particularly a cell surface receptor, a regulatory protein,such as a growth factor or other regulatory agent, or any other proteinor peptide that one desires to deliver to a cell in a living vertebrateand for which corresponding DNA or mRNA can be obtained.

[0043] In preferred embodiments, the polynucleotide is introduced intomuscle tissue; in other embodiments the polynucleotide is incorporatedinto tissuess of skin, brain, lung, liver, spleen or blood. Thepreparation is injected into the vertebrate by a variety of routes,which may be intradermally, subdermally, intrathecally, orintravenously, or it may be placed within cavities of the body. In apreferred embodiment, the polynucleotide is injected intramuscularly. Instill other embodiments, the preparation comprising the polynucleotideis impressed into the skin. Transdermal administration is alsocontemplated, as is inhalation.

[0044] In one preferred embodiment, the polynucleotide is DNA coding forboth a polypeptide and a polymerase for transcribing the DNA, and theDNA includes recognition sites for the polymerase and the injectablepreparation further includes a means for providing an initial quantityof the polymerase in the cell. The initial quantity of polymerase may bephysically present together with the DNA. Alternatively, it may beprovided by including mRNA coding therefor, which mRNA is translated bythe cell. In this embodiment of the invention, the DNA is preferably aplasmid. Preferably, the polymerase is phage T7 polymerase and therecognition site is a T7 origin of replication sequence.

[0045] In accordance with another aspect of the invention, there isprovided a method for treating a disease associated with the deficiencyor absence of a specific polypeptide in a vertebrate, comprising thesteps of obtaining an injectable preparation comprising apharmaceutically acceptable injectable carrier containing a nakedpolynucleotide coding for the specific polypeptide; introducing theinjectable preparation into a vertebrate and permitting thepolynucleotide to be incorporated into a cell, wherein the polypeptideis formed as the translation product of the polynucleotide, and wherebythe deficiency or absence of the polypeptide is compensated for. Inpreferred embodiments, the preparation is introduced into muscle tissueand the method is applied repetitively. The method is advantageouslyapplied where the deficiency or absence is due to a genetic defect. Thepolynucleotide is preferably a non-replicating DNA sequence; the DNAsequence may also be incorporated into a plasmid vector which comprisesan origin of replication.

[0046] In one of the preferred embodiments, the polynucleotide codes fora non-secreted polypeptide, and the polypeptide remains in situ.According to this embodiment, when the polynucleotide codes for thepolypeptide dystrophin, the method provides a therapy for Duchenne'ssyndrome; alternatively, when the polynucleotide codes for thepolypeptide phenylalanine hydroxylase, the method comprises a therapyfor phenylketonuria. In another preferred embodiment of the method, thepolynucleotide codes for a polypeptide which is secreted by the cell andreleased into the circulation of the vertebrate; in a particularlypreferred embodiment the polynucleotide codes for human growth hormone.

[0047] In yet another embodiment of the method, there is provided atherapy for hypercholesterolemia wherein a polynucleotide coding for areceptor associated with cholesterol homeostasis is introduced into aliver cell, and the receptor is expressed by the cell.

[0048] In accordance with another aspect of the present invention, thereis provided a method for immunizing a vertebrate, comprising the stepsof obtaining a preparation comprising an expressible polynucleotidecoding for an immunogenic translation product, and introducing thepreparation into a vertebrate wherein the translation product of thepolynucleotide is formed by a cell of the vertebrate, which elicits animmune response against the immunogen. In one embodiment of the method,the injectable preparation comprises a pharmaceutically acceptablecarrier containing an expressible polynucleotide coding for animmunogenic peptide, and on the introduction of the preparation into thevertebrate, the polynucleotide is incorporated into a cell of thevertebrate wherein an immunogenic translation product of thepolynucleotide is formed, which elicits an immune response against theimmunogen.

[0049] In an alternative embodiment, the preparation comprises one ormore cells obtained from the vertebrate and transfected in vitro withthe polynucleotide, whereby the polynucleotide is incorporated into saidcells, where an immunogenic translation product of the polynucleotide isformed, and whereby on the introduction of the preparation into thevertebrate, an immune response against the immunogen is elicited. In anyof the embodiments of the invention, the immunogenic product may besecreted by the cells, or it may be presented by a cell of thevertebrate in the context of the major histocompatibility antigens,thereby eliciting an immune response against the immunogen. The methodmay be practiced using non-dividing, differentiated cells from thevertebrates, which cells may be lymphocytes, obtained from a bloodsample; alternatively, it may be practiced using partiallydifferentiated skin fibroblasts which are capable of dividing. In apreferred embodiment, the method is practiced by incorporating thepolynucleotide coding for an immunogenic translation product into muscletissue.

[0050] The polynucleotide used for immunization is preferably an mRNAsequence, although a non-replicating DNA sequence may be used. Thepolynucleotide may be introduced into tissues of the body using theinjectable carrier alone; liposomal preparations are preferred formethods in which in vitro transfections of cells obtained from thevertebrate are carried out.

[0051] The carrier preferably is isotonic, hypotonic, or weaklyhypertonic, and has a relatively low ionic strength, such as provided bya sucrose solution. The preparation may further advantageously comprisea source of a cytokine which is incorporated into liposomes in the formof a polypeptide or as a polynucleotide.

[0052] The method may be used to selectively elicit a humoral immuneresponse, a cellular immune response, or a mixture of these. Inembodiments wherein the cell expresses major histocompatibility complexof Class I, and the immunogenic peptide is presented in the context ofthe Class I complex, the immune response is cellular and comprises theproduction of cytotoxic T-cells.

[0053] In one such embodiment, the immunogenic peptide is associatedwith a virus, is presented in the context of Class I antigens, andstimulates cytotoxic T-cells which are capable of destroying cellsinfected with the virus. A cytotoxic T-cell response may also beproduced according the method where the polynucleotide codes for atruncated viral antigen lacking humoral epitopes.

[0054] In another of these embodiments, the immunogenic peptide isassociated with a tumor, is presented in the context of Class Iantigens, and stimulates cytotoxic T cells which are capable ofdestroying tumor cells. In yet another embodiment wherein the injectablepreparation comprises cells taken from the animal and transfected invitro, the cells expressing major histocompatibility antigen of class Iand class II, and the immune response is both humoral and cellular andcomprises the production of both antibody and cytotoxic T-cells.

[0055] In another embodiment, there is provided a method of immunizing avertebrate, comprising the steps of obtaining a positively chargedliposome containing an expressible polynucleotide coding for animmunogenic peptide, and introducing the liposome into a vertebrate,whereby the liposome is incorporated into a monocyte, a macrophage, oranother cell, where an immunogenic translation product of thepolynucleotide is formed, and the product is processed and presented bythe cell in the context of the major histocompatibility complex, therebyeliciting an immune response against the immunogen. Again, thepolynucleotide is preferably mRNA, although DNA may also be used. And asbefore, the method may be practiced without the liposome, utilizing justthe polynucleotide in an injectable carrier.

[0056] The present invention also encompasses the use of DNA coding fora polypeptide and for a polymerase for transcribing the DNA, and whereinthe DNA includes recognition sites for the polymerase. The initialquantity of polymerase is provided by including mRNA coding therefor inthe preparation, which mRNA is translated by the cell. The mRNApreferably is provided with means for retarding its degradation in thecell. This can include capping the mRNA, circularizing the mRNA, orchemically blocking the 5′ end of the mRNA. The DNA used in theinvention may be in the form of linear DNA or may be a plasmid. EpisomalDNA is also contemplated. One preferred polymerase is phage T7 RNApolymerase and a preferred recognition site is a T7 RNA polymerasepromoter.

DETAILED DESCRIPTION OF THE INVENTION

[0057] The practice of the present invention requires obtaining nakedpolynucleotide operatively coding for a polypeptide for incorporationinto vertebrate cells. A polynucleotide operatively codes for apolypeptide when it has all the genetic information necessary forexpression by a target cell, such as promoters and the like. Thesepolynucleotides can be administered to the vertebrate by any method thatdelivers injectable materials to cells of the vertebrate, such as byinjection into the interstitial space of tissues such as muscles orskin, introduction into the circulation or into body cavities or byinhalation or insufflation. A naked polynucleotide is injected orotherwise delivered to the animal with a pharmaceutically acceptableliquid carrier. In preferred applications, the liquid carrier is aqueousor partly aqueous, comprising sterile, pyrogen-free water. The pH of thepreparation is suitably adjusted and buffered. The polynucleotide cancomprise a complete gene, a fragment of a gene, or several genes,together with recognition and other sequences necessary for expression.

[0058] In the embodiments of the invention that require use ofliposomes, for example, when the polynucleotide is to be associated witha liposome, it requires a material for forming liposomes, preferablycationic or positively charged liposomes, and requires that liposomalpreparations be made from these materials. With the liposomal materialin hand, the polynucleotide may advantageously be used to transfectcells in vitro for use as immunizing agents, or to administerpolynucleotides into bodily sites where liposomes may be taken up byphagocytic cells.

[0059] Polynucleotide Materials

[0060] The naked polynucleotide materials used according to the methodsof the invention comprise DNA and RNA sequences or DNA and RNA sequencescoding for polypeptides that have useful therapeutic applications. Thesepolynucleotide sequences are naked in the sense that they are free fromany delivery vehicle that can act to facilitate entry into the cell, forexample, the polynucleotide sequences are free of viral sequences,particularly any viral particles which may carry genetic information.They are similarly free from, or naked with respect to, any materialwhich promotes transfection, such as liposomal formulations, chargedlipids such as Lipofectin™ or precipitating agents such as CaPO₄.

[0061] The DNA sequences used in these methods can be those sequenceswhich do not integrate into the genome of the host cell. These may benon-replicating DNA sequences, or specific replicating sequencesgenetically engineered to lack the genome-integration ability.

[0062] The polynucleotide sequences of the invention are DNA or RNAsequences having a therapeutic effect after being taken up by a cell.Examples of polynucleotides that are themselves therapeutic areanti-sense DNA and RNA; DNA coding for an anti-sense RNA; or DNA codingfor tRNA or rRNA to replace defective or deficient endogenous molecules.The polynucleotides of the invention can also code for therapeuticpolypeptides. A polypeptide is understood to be any translation productof a polynucleotide regardless of size, and whether glycosylated or not.Therapeutic polypeptides include as a primary example, thosepolypeptides that can compensate for defective or deficient species inan animal, or those that act through toxic effects to limit or removeharmful cells from the body.

[0063] Therapeutic polynucleotides provided by the invention can alsocode for immunity-conferring polypeptides, which can act as endogenousimmunogens to provoke a humoral or cellular response, or both. Thepolynucleotides employed according to the present invention can alsocode for an antibody. In this regard, the term “antibody” encompasseswhole immunoglobulin of any class, chimeric antibodies and hybridantibodies with dual or multiple antigen or epitope specificities, andfragments, such as F(ab)₂, Fab′, Fab and the like, including hybridfragments. Also included within the meaning of “antibody” are conjugatesof such fragments, and so-called antigen binding proteins (single chainantibodies) as described, for example, in U.S. Pat. No. 4,704,692, thecontents of which are hereby incorporated by reference.

[0064] Thus, an isolated polynucleotide coding for variable regions ofan antibody can be introduced, in accordance with the present invention,to enable the treated subject to produce antibody in situ. Forillustrative methodology relating to obtaining antibody-encodingpolynucleotides, see Ward et al. Nature, 341:544-546 (1989); Gillies etal., Biotechnol. 7:799-804 (1989); and Nakatani et al., loc. cit.,805-810 (1989). The antibody in turn would exert a therapeutic effect,for example, by binding a surface antigen associated with a pathogen.Alternatively, the encoded antibodies can be anti-idiotypic antibodies(antibodies that bind other antibodies) as described, for example, inU.S. Pat. No. 4,699,880. Such anti-idiotypic antibodies could bindendogenous or foreign antibodies in a treated individual, thereby toameliorate or prevent pathological conditions associated with an immuneresponse, e.g., in the context of an autoimmune disease.

[0065] Polynucleotide sequences of the invention preferably code fortherapeutic or immunogenic polypeptides, and these sequences may be usedin association with other polynucleotide sequences coding for regulatoryproteins that control the expression of these polypeptides. Theregulatory protein can act by binding to genomic DNA so as to regulateits transcription; alternatively, it can act by binding to messenger RNAto increase or decrease its stability or translation efficiency.

[0066] The polynucleotide material delivered to the cells in vivo cantake any number of forms, and the present invention is not limited toany particular polynucleotide coding for any particular polypeptide.Plasmids containing genes coding for a large number of physiologicallyactive peptides and antigens or immunogens have been reported in theliterature and can be readily obtained by those of skill in the art.

[0067] Where the polynucleotide is to be DNA, promoters suitable for usein various vertebrate systems are well known. For example, for use inmurine systems, suitable strong promoters include RSV LTR, MPSV LTR,SV40 IEP, and metallothionein promoter. In humans, on the other hand,promoters such as CMV IEP may advantageously be used. All forms of DNA,whether replicating or non-replicating, which do not become integratedinto the genome, and which are expressible, are within the methodscontemplated by the invention.

[0068] With the availability of automated nucleic acid synthesisequipment, both DNA and RNA can be synthesized directly when thenucleotide sequence is known or by a combination of PCR cloning andfermentation. Moreover, when the sequence of the desired polypeptide isknown, a suitable coding sequence for the polynucleotide can beinferred.

[0069] When the polynucleotide is mRNA, it can be readily prepared fromthe corresponding DNA in vitro. For example, conventional techniquesutilize phage RNA polymerases SP6, T3, or T7 to prepare mRNA from DNAtemplates in the presence of the individual ribonucleosidetriphosphates. An appropriate phage promoter, such as a T7 origin ofreplication site is placed in the template DNA immediately upstream ofthe gene to be transcribed. Systems utilizing T7 in this manner are wellknown, and are described in the literature, e.g., in Current Protocolsin Molecular Biology, §3.8 (Vol.1 1988).

[0070] One particularly preferred method for obtaining the mRNA used inthe present invention is set forth in Examples 2-5. In general, however,it should be apparent that the pXGB plasmid or any similar plasmid thatcan be readily constructed by those of ordinary skill in the art can beused with a virtually unlimited number of cDNAs in practicing thepresent invention. Such plasmids may advantageously comprise a promoterfor a desired RNA polymerase, followed by a 5′ untranslated region, a 3′untranslated region, and a template for a poly A tract. There should bea unique restriction site between these 5′ and 3′ regions to facilitatethe insertion of any desired cDNA into the plasmid. Then, after cloningthe plasmid containing the desired gene, the plasmid is linearized bycutting in the polyadenylation region and is transcribed in vitro toform mRNA transcripts. These transcripts are preferably provided with a5′ cap, as demonstrated in Example 5. Alternatively, a 5′ untranslatedsequence such as EMC can be used which does not require a 5′ cap.

[0071] While the foregoing represents a preferred method for preparingthe mRNA, it will be apparent to those of skill in the art that manyalternative methods also exist. For example, the mRNA can be prepared incommercially-available nucleotide synthesis apparatus. Alternatively,mRNA in circular form can be prepared. Exonuclease-resistant RNAs suchas circular mRNA, chemically blocked mRNA, and mRNA with a 5′ cap arepreferred, because of their greater half-life in vivo.

[0072] In particular, one preferred mRNA is a self-circularizing mRNAhaving the gene of interest preceded by the 5′ untranslated region ofpolio virus. It has been demonstrated that circular mRNA has anextremely long half-life (Harland & Misher, Development 102: 837-852(1988)) and that the polio virus 5′ untranslated region can promotetranslation of mRNA without the usual 5′ cap (Pelletier & Sonnenberg,Nature 334:320-325 (1988), hereby incorporated by reference).

[0073] This material may be prepared from a DNA template that isself-splicing and generates circular “lariat” mRNAs, using the method ofBeen & Cech, Cell 47:206-216 (1986) (hereby incorporated by reference).We modify that template by including the 5′ untranslated region of thepolio virus immediately upstream of the gene of interest, following theprocedure of Maniatis, T. et al. MOLECULAR CLONING: A LABORATORY MANUAL,Cold Spring Harbor, N.Y. (1982).

[0074] In addition, the present invention includes the use of mRNA thatis chemically blocked at the 5′ and/or 3′ end to prevent access byRNAse. (This enzyme is an exonuclease and therefore does not cleave RNAin the middle of the chain.) Such chemical blockage can substantiallylengthen the half life of the RNA in vivo. Two agents which may be usedto modify RNA are available from Clonetech Laboratories, Inc., PaloAlto, Calif. C2 AminoModifier (Catalog #5204-1) and Amino-7-dUTP(Catalog #K1022-1). These materials add reactive groups to the RNA.After introduction of either of these agents onto an RNA molecule ofinterest, an appropriate reactive substituent can be linked to the RNAaccording to the manufacturer's instructions. By adding a group withsufficient bulk, access to the chemically modified RNA by RNAse can beprevented.

[0075] Transient Gene Therapy

[0076] Unlike gene therapies proposed in the past, one major advantageof the present invention is the transitory nature of the polynucleotidesynthesis in the cells. (We refer to this as reversible gene therapy, orTGT.) With mRNA introduced according to the present invention, theeffect will generally last about one day. Also, in marked contrast togene therapies proposed in the past, mRNA does not have to penetrate thenucleus to direct protein synthesis; therefore, it should have nogenetic liability.

[0077] In some situations, however, a more prolonged effect may bedesired without incorporation of the exogenous polynucleic acid into thegenome of the host organism. In order to provide such an effect, apreferred embodiment of the invention provides introducing a DNAsequence coding for a specific polypeptide into the cell. We have found,according to the methods of the invention, that non-replicating DNAsequences can be introduced into cells to provide production of thedesired polypeptide for periods of about up to six months, and we haveobserved no evidence of integration of the DNA sequences into the genomeof the cells. Alternatively, an even more prolonged effect can beachieved by introducing the DNA sequence into the cell by means of avector plasmid having the DNA sequence inserted therein. Preferably, theplasmid further comprises a replicator. Such plasmids are well known tothose skilled in the art, for example, plasmid pBR322, with replicatorpMB1, or plasmid pMK16, with replicator ColE1 (Ausubel, CurrentProtocols in Molecular Biology, John Wiley and Sons, New York (1988)§II:1.5.2.

[0078] Results of studies of the time course of expression of DNA andmRNA introduced into muscle cells as described in Examples 1 and 13indicate that mRNA expression is more rapid, although shorter induration than DNA expression. An immediate and long lived geneexpression can be achieved by administering to the cell a liposomalpreparation comprising both DNA and an RNA polymerase, such as the phagepolymerases T7, T3, and SP6. The liposome also includes an initialsource of the appropriate RNA polymerase, by either including the actualenzyme itself, or alternatively, an mRNA coding for that enzyme. Whenthe liposome is introduced into the organism, it delivers the DNA andthe initial source of RNA polymerase to the cell. The RNA polymerase,recognizing the promoters on the introduced DNA, transcribes both genes,resulting in translation products comprising more RNA polymerase and thedesired polypeptide. Production of these materials continues until theintroduced DNA (which is usually in the form of a plasmid) is degraded.In this manner, production of the desired polypeptide in vivo can beachieved in a few hours and be extended for one month or more.

[0079] Although not limited to the treatment of genetic disease, themethods of the invention can accordingly be appropriately applied totreatment strategies requiring delivery and functional expression ofmissing or defective genes.

[0080] The polynucleotides may be delivered to the interstitial space oftissues of the animal body, including those of muscle, skin, brain,lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellular,fluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. We have discovered that in vivo muscle cells areparticularly competent in their ability to take up and expresspolynucleotides. This ability may be due to the singular tissuearchitecture of muscle, comprising multinucleated cells, sarcoplasmicreticulum, and transverse tubular system. Polynucleotides may enter themuscle through the transverse tubular system, which containsextracellular fluid and extends deep into the muscle cell. It is alsopossible that the polynucleotides enter damaged muscle cells which thenrecover.

[0081] Muscle is also advantageously used as a site for the delivery andexpression of polynucleotides in a number of therapeutic applicationsbecause animals have a proportionately large muscle mass which isconveniently accessed by direct injection through the skin; for thisreason, a comparatively large dose of polynucleotides can be depositedin muscle by multiple injections, and repetitive injections, to extendtherapy over long periods of time, are easily performed and can becarried out safely and without special skill or devices.

[0082] Muscle tissue can be used as a site for injection and expressionof polynucleotides in a set of general strategies, which are exemplaryand not exhaustive. First, muscle disorders related to defective orabsent gene products can be treated by introducing polynucleotidescoding for a non-secreted gene product into the diseased muscle tissue.In a second strategy, disorders of other organs or tissues due to theabsence of a gene product, and which results in the build-up of acirculating toxic metabolite can be treated by introducing the specifictherapeutic polypeptide into muscle tissue where the non-secreted geneproduct is expressed and clears the circulating metabolite. In a thirdstrategy, a polynucleotide coding for an secretable therapeuticpolypeptide can be injected into muscle tissue from where thepolypeptide is released into the circulation to seek a metabolic target.This use is demonstrated in the expression of growth hormone geneinjected into muscle, Example 18. Certain DNA segments, are known toserve as “signals” to direct secretion (Wickner, W. T. and H. F. Lodish,Science 230:400-407 (1985), and these may be advantageously employed.Finally, in immunization strategies, muscle cells may be injected withpolynucleotides coding for immunogenic peptides, and these peptides willbe presented by muscle cells in the context of antigens of the majorhistocompatibility complex to provoke a selected immune response againstthe immunogen.

[0083] Tissues other than those of muscle, and having a less efficientuptake and expression of injected polynucleotides, may nonetheless beadvantageously used as injection sites to produce therapeuticpolypeptides or polynucleotides under certain conditions. One suchcondition is the use of a polynucleotide to provide a polypeptide whichto be effective must be present in association with cells of a specifictype; for example, the cell surface receptors of liver cells associatedwith cholesterol homeostasis. (Brown and Goldstein, Science 232:34-47(1986)). In this application, and in many others, such as those in whichan enzyme or hormone is the gene product, it is not necessary to achievehigh levels of expression in order to effect a valuable therapeuticresult.

[0084] One application of TGT is in the treatment of muscular dystrophy.The genetic basis of the muscular dystrophies is just beginning to beunraveled. The gene related to Duchenne/Becker muscular dystrophy hasrecently been cloned and encodes a rather large protein, termeddystrophin. Retroviral vectors are unlikely to be useful, because theycould not accommodate the rather large size of the cDNA (about 13 kb)for dystrophin. Very recently reported work is centered on transplantingmyoblasts, but the utility of this approach remains to be determined.Clearly, an attractive approach would be to directly express thedystrophin gene within the muscle of patients with Duchennes. Since mostpatients die from respiratory failure, the muscles involved withrespiration would be a primary target.

[0085] Another application is in the treatment of cystic fibrosis. Thegene for cystic fibrosis was recently identified (Goodfellow, P. Nature,341(6238):102-3 (Sep. 14, 1989); Rommens, J. et al. Science,245(4922):1059-1065 (Sep. 8, 1989); Beardsley, T. et al., ScientificAmerican, 261(5):28-30 (1989). Significant amelioration of the symptomsshould be attainable by the expression of the dysfunctional proteinwithin the appropriate lung cells. The bronchial epithelial cells arepostulated to be appropriate target lung cells and they could beaccessible to gene transfer following instillation of genes into thelung. Since cystic fibrosis is an autosomal recessive disorder one wouldneed to achieve only about 5% of normal levels of the cystic fibrosisgene product in order to significantly ameliorate the pulmonarysymptoms.

[0086] Biochemical genetic defects of intermediary metabolism can alsobe treated by TGT. These diseases include phenylketonuria, galactosemia,maple-syrup urine disease, homocystinuria, propionic acidemia,methylmalonic acidemia, and adenosine deaminase deficiency. Thepathogenesis of disease in most of these disorders fits thephenylketonuria (PKU) model of a circulating toxic metabolite. That is,because of an enzyme block, a biochemical, toxic to the body,accumulates in body fluids. These disorders are ideal for gene therapyfor a number of reasons. First, only 5% of normal levels of enzymeactivity would have to be attained in order to significantly clearenough of the circulating toxic metabolite so that the patient issignificantly improved. Second, the transferred gene could most often beexpressed in a variety of tissues and still be able to clear the toxicbiochemical.

[0087] Reversible gene therapy can also be used in treatment strategiesrequiring intracytoplasmic or intranuclear protein expression. Someproteins are known that are capable of regulating transcription bybinding to specific promoter regions on nuclear DNA. Other proteins bindto RNA, regulating its degradation, transport from the nucleus, ortranslation efficiency. Proteins of this class must be deliveredintracellularly for activity. Extracellular delivery of recombinanttranscriptional or translational regulatory proteins would not beexpected to have biological activity, but functional delivery of the DNAor RNA by TGT would be active. Representative proteins of this type thatwould benefit from TGT would include NEF, TAT, steroid receptor and theretinoid receptor.

[0088] Gene therapy can be used in a strategy to increase the resistanceof an AIDS patient to HIV infection. Introducing an AIDS resistancegene, such as, for example, the NEF gene or the soluble CD4 gene toprevent budding, into an AIDS patient's T cells will render his T cellsless capable of producing active AIDS virus, thus sparing the cells ofthe immune system and improving his ability to mount a T cell dependentimmune response. Thus, in accordance with the invention, a population ofthe AIDS patient's own T cells is isolated from the patient's blood.These cells are then transfected in vitro and then reintroduced backinto the patient's blood. The virus-resistant cells will have aselective advantage over the normal cells, and eventually repopulate thepatient's lymphatic system. DNA systemic delivery to macrophages orother target cells can be used in addition to the extracorporealtreatment strategy. Although this strategy would not be expected toeradicate virus in the macrophage reservoir, it will increase the levelof T cells and improve the patient's immune response.

[0089] In all of the systemic strategies presented herein, an effectiveDNA or mRNA dosage will generally be in the range of from about 0.05μg/kg to about 50 mg/kg, usually about 0.005-5 mg/kg. However, as willbe appreciated, this dosage will vary in a manner apparent to those ofskill in the art according to the activity of the peptide coded for bythe DNA or mRNA and the particular peptide used. For delivery ofadenosine deaminase to mice or humans, for example, adequate levels oftranslation are achieved with a DNA or mRNA dosage of about 0.5 to 5mg/kg. See Example 10. From this information, dosages for other peptidesof known activity can be readily determined.

[0090] Diseases which result from deficiencies of critical proteins maybe appropriately treated by introducing into specialized cells, DNA ormRNA coding for these proteins. A variety of growth factors such asnerve growth factor and fibroblast growth factor have been shown toaffect neuronal cell survival in animal models of Alzheimer's disease.In the aged rat model, NGF infusions have reversed the loss ofcholinergic neurons. In the fimbria-fornix lesion rat, NGF infusions orsecretion from genetically-modified fibroblasts have also avoided theloss of cholinergic function. Cholinergic activity is diminished inpatients with Alzheimer's. The expression within the brain of transducedgenes expressing growth factors could reverse the lost of function ofspecific neuronal groups.

[0091] Introduction of DNA or mRNA by transfection of the gene forneuronal growth factor into cells lining the cranial cavity can be usedin accordance with the present invention in the treatment of Alzheimer'sdisease. In particular, the present invention treats this disease byintracranial injection of from about 10 μg to about 100 μg of DNA ormRNA into the parenchyma through use of a stereotaxic apparatus.Specifically, the injection is targeted to the cholinergic neurons inthe medial septum. The DNA or mRNA injection is repeated every 1-3 daysfor 5′ capped, 3′ polyadenylated mRNA, and every week to 21 days forcircular mRNA, and every 30 to 60 days for DNA. Injection of DNA inaccordance with the present invention is also contemplated. DNA would beinjected in corresponding amounts; however, frequency of injection wouldbe greatly reduced. Episomal DNA, for example, could be active for anumber of months, and reinjection would only be necessary upon notableregression by the patient.

[0092] In addition, the enzymes responsible for neurotransmittersynthesis could be expressed from transduced genes. For example, thegene for choline acetyl transferase could be expressed within the braincells (neurons or glial) of specific areas to increase acetylcholinelevels and improve brain function.

[0093] The critical enzymes involved in the synthesis of otherneurotransmitters such as dopamine, norepinephrine, and GABA have beencloned and available. The critical enzymes could be locally increased bygene transfer into a localized area of the brain. The increasedproductions of these and other neurotransmitters would have broadrelevance to manipulation of localized neurotransmitter function andthus to a broad range of brain disease in which disturbedneurotransmitter function plays a crucial role. Specifically, thesediseases could include schizophrenia and manic-depressive illnesses andParkinson's Disease. It is well established that patients withParkinson's suffer from progressively disabled motor control due to thelack of dopamine synthesis within the basal ganglia. The rate limitingstep for dopamine synthesis is the conversion of tyrosine to L-DOPA bythe enzyme, tyrosine hydroxylase. L-DOPA is then converted to dopamineby the ubiquitous enzyme, DOPA decarboxylase. That is why thewell-established therapy with L-DOPA is effective (at least for thefirst few years of treatment). Gene therapy could accomplish the similarpharmacologic objective by expressing the genes for tyrosine hydroxylaseand possible DOPA decarboxylase as well. Tyrosine is readily availablewithin the CNS.

[0094] The genetic form of alpha-1-antitrypsin deficiency can result inboth liver and lung disease. The liver disease, which is less common, iscaused by the accumulation of an abnormal protein and would be lessamenable to gene therapy. The pulmonary complications, however, would beamenable to the increased expression of alpha-1-antitrypsin within thelung. This should prevent the disabling and eventually lethal emphysemafrom developing.

[0095] Alpha-1-antitrypsin deficiency also occurs in tobacco smokerssince tobacco smoke decreases alpha-1-antitrypsin activity and thusserine protease activity that leads to emphysema. In addition, somerecent data links tobacco smoke's anti-trypsin effect to aneurysms ofthe aorta. Aneurysms would also be preventable by raising blood levelsof anti-1-antitrypsin since this would decrease protease activity thatleads to aneurysms.

[0096] Patients with degenerative disease of the lung could also benefitfrom the expression of enzymes capable of removing other toxicmetabolites which tend to accumulate in diseased lung tissue. Superoxidedismutase and catalase could be delivered by TGT to ameliorate theseproblems.

[0097] TGT can be used in treatment strategies requiring the delivery ofcell surface receptors. It could be argued that there is no need todecipher methodology for functional in vivo delivery of genes. There is,after all, an established technology for the synthesis and large scaleproduction of proteins, and proteins are the end product of geneexpression. This logic applies for many protein molecules which actextracellularly or interact with cell surface receptors, such as tissueplasminogen activator (TPA), growth hormone, insulin, interferon,granulocyte-macrophage colony stimulating factor (GMCSF), erythropoietin(EPO), etc. However, the drug delivery problems associated with properlydelivering a recombinant cell surface receptor to be inserted in theplasma membrane of its target cell in the proper orientation for afunctional receptor have hithertofore appeared intractable.

[0098] When DNA or RNA coding for a cell surface receptor is deliveredintracellularly in accordance with the present invention, the resultingprotein can be efficiently and functionally expressed on the target cellsurface. If the problem of functional delivery of recombinant cellsurface receptors remains intractable, then the only way of approachingthis therapeutic modality will be through gene delivery. Similar logicfor nuclear or cytoplasmic regulation of gene expression applies tonuclear regulatory factor bound to DNA to regulate (up or down) RNAtranscription and to cytoplasmic regulatory factors which bind to RNA toincrease or decrease translational efficiency and degradation. TGT couldin this way provide therapeutic strategies for the treatment of cysticfibrosis, muscular dystrophy and hypercholesterolemia.

[0099] Elevated levels of cholesterol in the blood may be reduced inaccordance with the present invention by supplying mRNA coding for theLDL surface receptor to hepatocytes. A slight elevation in theproduction of this receptor in the liver of patients with elevated LDLwill have significant therapeutic benefits. Therapies based on systemicadministration of recombinant proteins are not able to compete with thepresent invention, because simply administering the recombinant proteincould not get the receptor into the plasma membrane of the target cells.The receptor must be properly inserted into the membrane in order toexert its biological effect. It is not usually necessary to regulate thelevel of receptor expression; the more expression the better. Thissimplifies the molecular biology involved in preparation of the mRNA foruse in the present invention. For example, lipid/DNA or RNA complexescontaining the LDL receptor gene may be prepared and supplied to thepatient by repetitive I.V. injections. The lipid complexes will be takenup largely by the liver. Some of the complexes will be taken up byhepatocytes. The level of LDL receptor in the liver will increasegradually as the number of injections increases. Higher liver LDLreceptor levels will lead to therapeutic lowering of LDL andcholesterol. An effective mRNA dose will generally be from about 0.1 toabout 5 mg/kg.

[0100] Other examples of beneficial applications of TGT include theintroduction of the thymidine kinase gene into macrophages of patientsinfected with the HIV virus. Introduction of the thymidine kinase geneinto the macrophage reservoir will render those cells more capable ofphosphorylating AZT. This tends to overcome their resistance to AZTtherapy, making AZT capable of eradicating the HIV reservoir inmacrophages. Lipid/DNA complexes containing the thymidine kinase genecan be prepared and administered to the patient through repetitiveintravenous injections. The lipid complexes will be taken up largely bythe macrophage reservoir leading to elevated levels of thymidine kinasein the macrophages. This will render the AZT resistant cells subject totreatment with AZT. The thymidine kinase therapy can also be focused byputting the thymidine kinase gene under the control of the HTLV IIIpromoter. According to this strategy, the thymidine kinase would only besynthesized on infection of the cell by HIV virus, and the production ofthe tat protein which activates the promoter. An analogous therapy wouldsupply cells with the gene for diphtheria toxin under the control of thesame HTLV III promoter, with the lethal result occurring in cells onlyafter HIV infection.

[0101] These AIDS patients could also be treated by supplying theinterferon gene to the macrophages according to the TGT method.Increased levels of localized interferon production in macrophages couldrender them more resistant to the consequences of HIV infection. Whilelocal levels of interferon would be high, the overall systemic levelswould remain low, thereby avoiding the systemic toxic effects like thoseobserved after recombinant interferon administration. Lipid/DNA or RNAcomplexes containing the interferon gene can be prepared andadministered to the patient by repetitive intravenous injections. Thelipid complexes will be taken up largely by the macrophage reservoirleading to elevated localized levels of interferon in the macrophages.This will render them less susceptible to HIV infection.

[0102] Various cancers may be treated using TGT by supplying adiphtheria toxin gene on a DNA template with a tissue specific enhancerto focus expression of the gene in the cancer cells. Intracellularexpression of diphtheria toxin kills cells. These promoters could betissue-specific such as using a pancreas-specific promoter for thepancreatic cancer. A functional diphtheria toxin gene delivered topancreatic cells could eradicate the entire pancreas. This strategycould be used as a treatment for pancreatic cancer. The patients wouldhave no insurmountable difficulty surviving without a pancreas. Thetissue specific enhancer would ensure that expression of diphtheriatoxin would only occur in pancreatic cells. DNA/lipid complexescontaining, the diphtheria toxin gene under the control of a tissuespecific enhancer would be introduced directly into a cannulated arteryfeeding the pancreas. The infusion would occur on some dosing schedulefor as long as necessary to eradicate the pancreatic tissue. Otherlethal genes besides diphtheria toxin could be used with similar effect,such as genes for ricin or cobra venom factor or enterotoxin.

[0103] Also, one could treat cancer by using a cell-cycle specificpromoter that would only kill cells that are rapidly cycling (dividing)such as cancer cells. Cell-cycle specific killing could also beaccomplished by designing mRNA encoding killer proteins that are stableonly in cycling cells (i.e. histone mRNA that is only stable during Sphase). Also, one could use developmental-specific promoters such as theuse of alpha-fetoprotein that is only expressed in fetal liver cells andin hepatoblastoma cells that have dedifferentiated into a more fetalstate.

[0104] One could also treat specialized cancers by the transfer of genessuch as the retinoblastoma gene (and others of that family) thatsuppress the cancer properties of certain cancers.

[0105] The TGT strategy can be used to provide a controlled, sustaineddelivery of peptides. Conventional drugs, as well as recombinant proteindrugs, can benefit from controlled release devices. The purpose of thecontrolled release device is to deliver drugs over a longer time period,so that the number of doses required is reduced. This results inimprovements in patient convenience and compliance. There are a widevariety of emerging technologies that are intended to achieve controlledrelease.

[0106] TGT can be used to obtain controlled delivery of therapeuticpeptides. Regulated expression can be obtained by using suitablepromoters, including cell-specific promoters. Suitable peptidesdelivered by the present invention include, for example, growth hormone,insulin, interleukins, interferons, GMCSF, EPO, and the like. Dependingon the specific application, the DNA or an RNA construct selected can bedesigned to result in a gene product that is secreted from the injectedcells and into the systemic circulation.

[0107] TGT can also comprise the controlled delivery of therapeuticpolypeptides or peptides which is achieved by including with thepolynucleotide to be expressed in the cell, an additional polynucleotidewhich codes for a regulatory protein which controls processes oftranscription and translation. These polynucleotides comprise thosewhich operate either to up regulate or down regulate polypeptideexpression, and exert their effects either within the nucleus or bycontrolling protein translation events in the cytoplasm.

[0108] The T7 polymerase gene can be used in conjunction with a gene ofinterest to obtain longer duration of effect of TGT. Episomal DNA suchas that obtained from the origin of replication region for the EpsteinBarr virus can be used, as well as that from other origins ofreplication which are functionally active in mammalian cells, andpreferably those that are active in human cells. This is a way to obtainexpression from cells after many cell divisions, without riskingunfavorable integration events that are common to retrovirus vectors.Controlled release of calcitonin could be obtained if a calcitonin geneunder the control of its own promoter could be functionally introducedinto some site, such as liver or skin. Cancer patients withhypercalcemia would be a group to whom this therapy could be applied.

[0109] Other gene therapies using TGT can include the use of apolynucleotide that has a therapeutic effect without being translatedinto a polypeptide. For example, TGT can be used in the delivery ofanti-sense polynucleotides for turning off the expression of specificgenes. Conventional anti-sense methodology suffers from poor efficacy,in part, because the oligonucleotide sequences delivered are too short.With TGT, however, full length anti-sense sequences can be delivered aseasily as short oligomers. Anti-sense polynucleotides can be DNA or RNAmolecules that themselves hybridize to (and, thereby, preventtranscription or translation of) an endogenous nucleotide sequence.Alternatively, an anti-sense DNA may encode an RNA the hybridizes to anendogenous sequence, interfering with translation. Other uses of TGT inthis vein include delivering a polynucleotide that encodes a tRNA orrRNA to replace a defective or deficient endogenous tRNA or rRNA, thepresence of which causes the pathological condition.

[0110] Cell-specific promoters can also be used to permit expression ofthe gene only in the target cell. For example, certain genes are highlypromoted in adults only in particular types of tumors. Similarly,tissue-specific promoters for specialized tissue, e.g., lens tissue ofthe eye, have also been identified and used in heterologous expressionsystems.

[0111] Beyond the therapies described, the method of the invention canbe used to deliver polynucleotides to animal stock to increaseproduction of milk in dairy cattle or muscle mass in animals that areraised for meat.

[0112] DNA and mRNA Vaccines

[0113] According to the methods of the invention, both expressible DNAand mRNA can be delivered to cells to form therein a polypeptidetranslation product. If the nucleic acids contain the proper controlsequences, they will direct the synthesis of relatively large amounts ofthe encoded protein. When the DNA and mRNA delivered to the cells codesfor an immunizing peptide, the methods can be applied to achieveimproved and more effective immunity against infectious agents,including intracellular viruses, and also against tumor cells.

[0114] Since the immune systems of all vertebrates operate similarly,the applications described can be implemented in all vertebrate systems,comprising mammalian and avian species, as well as fish.

[0115] The methods of the invention may be applied by direct injectionof the polynucleotide into cells of the animal in vivo, or by in vitrotransfection of some of the animal cells which are then re-introducedinto the animal body. The polynucleotides may be delivered to variouscells of the animal body, including muscle, skin, brain, lung, liver,spleen, or to the cells of the blood. Delivery of the polynucleotidesdirectly in vivo is preferably to the cells of muscle or skin. Thepolynucleotides may be injected into muscle or skin using an injectionsyringe. They may also be delivered into muscle or skin using a vaccinegun.

[0116] It has recently been shown that cationic lipids can be used tofacilitate the transfection of cells in certain applications,particularly in vitro transfection. Cationic lipid based transfectiontechnology is preferred over other methods; it is more efficient andconvenient than calcium phosphate, DEAE dextran or electroporationmethods, and retrovirus mediated transfection, as discussed previously,can lead to integration events in the host cell genome that result inoncogene activation or other undesirable consequences. The knowledgethat cationic lipid technology works with messenger RNA is a furtheradvantage to this approach because RNA is turned over rapidly byintracellular nucleases and is not integrated into the host genome. Atransfection system that results in high levels of reversible expressionis preferred to alternative methodology requiring selection andexpansion of stably transformed clones because many of the desiredprimary target cells do not rapidly divide in culture.

[0117] The ability to transfect cells at high efficiency with cationicliposomes provides an alternative method for immunization. The gene foran antigen is introduced in to cells which have been removed from ananimal. The transfected cells, now expressing the antigen, arereinjected into the animal where the immune system can respond to the(now) endogenous antigen. The process can possibly be enhanced bycoinjection of either an adjuvant or lymphokines to further stimulatethe lymphoid cells.

[0118] Vaccination with nucleic acids containing a gene for an antigenmay also provide a way to specifically target the cellular immuneresponse. Cells expressing proteins which are secreted will enter thenormal antigen processing pathways and produce both a humoral andcytotoxic response. The response to proteins which are not secreted ismore selective. Non-secreted proteins synthesized in cells expressingonly class I MHC molecules are expected to produce only a cytotoxicvaccination. Expression of the same antigen in cells bearing both classI and class II molecules may produce a more vigorous response bystimulating both cytotoxic and helper T cells. Enhancement of the immuneresponse may also be possible by injecting the gene for the antigenalong with a peptide fragment of the antigen. The antigen is presentedvia class I MHC molecules to the cellular immune system while thepeptide is presented via class II MHC molecules to stimulate helper Tcells. In any case, this method provides a way to stimulate and modulatethe immune response in a way which has not previously been possible.

[0119] A major disadvantage of subunit vaccines is that glycoproteinantigens are seldom modified correctly in the recombinant expressionsystems used to make the antigens. Introducing the gene for aglycoprotein antigen will insure that the protein product issynthesized, modified and processed in the same species and cells thatthe pathogen protein would be. Thus, the expression of a gene for ahuman viral glycoprotein will contain the correct complement of sugarresidues. This is important because it has been demonstrated that asubstantial component of the neutralizing antibodies in some viralsystems are directed at carbohydrate epitopes.

[0120] Any appropriate antigen which is a candidate for an immuneresponse, whether humoral or cellular, can be used in its nucleic acidform. The source of the cells could be fibroblasts taken from anindividual which provide a convenient source of cells expressing onlyclass I MHC molecules. Alternatively, peripheral blood cells can berapidly isolated from whole blood to provide a source of cellscontaining both class I and class II MHC proteins. They could be furtherfractionated into B cells, helper T cells, cytotoxic T cells ormacrophage/monocyte cells if desired. Bone marrow cells can provide asource of less differentiated lymphoid cells. In all cases the cell willbe transfected either with DNA containing a gene for the antigen or bythe appropriate capped and polyadenylated mRNA transcribed from thatgene or a circular RNA, chemically modified RNA, or an RNA which doesnot require 5′ capping. The choice of the transfecting nucleotide maydepend on the duration of expression desired. For vaccination purposes,a reversible expression of the immunogenic peptide, as occurs on mRNAtransfection, is preferred. Transfected cells are injected into theanimal and the expressed proteins will be processed and presented to theimmune system by the normal cellular pathways.

[0121] Such an approach has been used to produce cytotoxic immunity inmodel systems in mice. Cell lines, malignant continuously growing cells,can be stably transformed with DNA. When cells are injected intoanimals, they induce cellular immunity to the expressed antigen. Thecationic lipid delivery system will allow this approach to be extendedto normal, non-malignant cells taken from a patient.

[0122] There are several applications to this approach of targetingcellular immunity. The first is vaccination against viruses in whichantibodies are known to be required or to enhanced viral infection.There are two strategies that can be applied here. One can specificallytarget the cellular pathway during immunization thus eliminating theenhancing antibodies. Alternatively one can vaccinate with the gene fora truncated antigen which eliminate the humoral epitomes which enhanceinfectivity.

[0123] The use of DNA or mRNA vaccine therapy could similarly provide ameans to provoke an effective cytotoxic T-cell response to weaklyantigenic tumors. We propose, for example, that if a tumor-specificantigen were expressed by mRNA inside a cell in an already processedform, and incorporated directly into the Class I molecules on the cellsurface, a cytotoxic T cell response would be elicited.

[0124] A second application is that this approach provides a method totreat latent viral infections. Several viruses (for example, HepatitisB, HIV and members of the Herpes virus group) can establish latentinfections in which the virus is maintained intracellularly in aninactive or partially active form. There are few ways of treating suchan infections. However, by inducing a cytolytic immunity against alatent viral protein, the latently infected cells will be targeted andeliminated.

[0125] A related application of this approach is to the treatment ofchronic pathogen infections. There are numerous examples of pathogenswhich replicate slowly and spread directly from cell to cell. Theseinfections are chronic, in some cases lasting years or decades. Examplesof these are the slow viruses (e.g. Visna), the Scrapie agent and HIV.One can eliminate the infected cells by inducing an cellular response toproteins of the pathogen.

[0126] Finally, this approach may also be applicable to the treatment ofmalignant disease. Vaccination to mount a cellular immune response to aprotein specific to the malignant state, be it an activated oncogene, afetal antigen or an activation marker, will result in the elimination ofthese cells.

[0127] The use of DNA/mRNA vaccines could in this way greatly enhancethe immunogenicity of certain viral proteins, and cancer-specificantigens, that normally elicit a poor immune response. The mRNA vaccinetechnique should be applicable to the induction of cytotoxic T cellimmunity against poorly immunogenic viral proteins from the Herpesviruses, non-A, non-B hepatitis, and HIV, and it would avoid the hazardsand difficulties associated with in vitro propagation of these viruses.For cell surface antigens, such as viral coat proteins (e.g., HIVgp120), the antigen would be expressed on the surface of the target cellin the context of the major histocompatibility complex (MHC), whichwould be expected to result in a more appropriate, vigorous andrealistic immune response. It is this factor that results in the moreefficacious immune responses frequently observed with attenuated virusvaccines. Delivery of a single antigen gene by TGT would be much saferthan attenuated viruses, which can result in a low frequency of diseasedue to inadequate attenuation.

[0128] There is an additional advantage of TGT which can be exploitedduring the vaccine development phase. One of the difficulties withvaccine development is the requirement to screen different structuralvariants of the antigen, for the optimal immune response. If the variantis derived from a recombinant source, the protein usually must beexpressed and purified before it can be tested for antigenicity. This isa laborious and time consuming process. With in vitro mutagenesis, it ispossible to obtain and sequence numerous clones of a given antigen. Ifthese antigen can be screened for antigenicity at the DNA or RNA levelby TGT, the vaccine development program could be made to proceed muchfaster.

[0129] Finally, in the case of the DNA/mRNA vaccines, the proteinantigen is never exposed directly to serum antibody, but is alwaysproduced by the transfected cells themselves following translation ofthe mRNA. Hence, anaphylaxis should not be a problem. Thus, the presentinvention permits the patient to be immunized repeatedly without thefear of allergic reactions. The use of the DNA/mRNA vaccines of thepresent invention makes such immunization possible.

[0130] One can easily conceive of ways in which this technology can bemodified to enhance still further the immunogenicity of antigens. T cellimmunization can be augmented by increasing the density of Class I andClass II histocompatibility antigens on the macrophage or other cellsurface and/or by inducing the transfected cell to release cytokinesthat promote lymphocyte proliferation. To this end, one may incorporatein the same liposomes that contain mRNA for the antigen, other mRNAspecies that encode interferons or interleukin-1. These cytokines areknown to enhance macrophage activation. Their systemic use has beenhampered because of side effects. However, when encapsulated in mRNA,along with mRNA for antigen, they should be expressed only by thosecells that co-express antigen. In this situation, the induction of Tcell immunity can be enhanced greatly.

[0131] Therapeutic Formulations

[0132] Polynucleotide salts: Administration of pharmaceuticallyacceptable salts of the polynucleotides described herein is includedwithin the scope of the invention. Such salts may be prepared frompharmaceutically acceptable non-toxic bases including organic bases andinorganic bases. Salts derived from inorganic bases include sodium,potassium, lithium, ammonium, calcium, magnesium, and the like. Saltsderived from pharmaceutically acceptable organic non-toxic bases includesalts of primary, secondary, and tertiary amines, basic amino acids, andthe like. For a helpful discussion of pharmaceutical salts, see S. M.Berge et al., Journal of Pharmaceutical Sciences 66:1-19 (1977) thedisclosure of which is hereby incorporated by reference.

[0133] Polynucleotides for injection, a preferred route of delivery, maybe prepared in unit dosage form in ampules, or in multidose containers.The polynucleotides may be present in such forms as suspensions,solutions, or emulsions in oily or preferably aqueous vehicles.Alternatively, the polynucleotide salt may be in lyophilized form forreconstitution, at the time of delivery, with a suitable vehicle, suchas sterile pyrogen-free water. Both liquid as well as lyophilized formsthat are to be reconstituted will comprise agents, preferably buffers,in amounts necessary to suitably adjust the pH of the injected solution.For any parenteral use, particularly if the formulation is to beadministered intravenously, the total concentration of solutes should becontrolled to make the preparation isotonic, hypotonic, or weaklyhypertonic. Nonionic materials, such as sugars, are preferred foradjusting tonicity, and sucrose is particularly preferred. Any of theseforms may further comprise suitable formulatory agents, such as starchor sugar, glycerol or saline. The compositions per unit dosage, whetherliquid or solid, may contain from 0.1% to 99% of polynucleotidematerial.

[0134] The units dosage ampules or multidose containers, in which thepolynucleotides are packaged prior to use, may comprise an hermeticallysealed container enclosing an amount of polynucleotide or solutioncontaining a polynucleotide suitable for a pharmaceutically effectivedose thereof, or multiples of an effective dose. The polynucleotide ispackaged as a sterile formulation, and the hermetically sealed containeris designed to preserve sterility of the formulation until use.

[0135] The container in which the polynucleotide is packaged is labeled,and the label bears a notice in the form prescribed by a governmentalagency, for example the Food and Drug Administration, which notice isreflective of approval by the agency under Federal law, of themanufacture, use, or sale of the polynucleotide material therein forhuman administration.

[0136] Federal law requires that the use of pharmaceutical agents in thetherapy of humans be approved by an agency of the Federal government.Responsibility for enforcement is the responsibility of the Food andDrug Administration, which issues appropriate regulations for securingsuch approval, detailed in 21 U.S.C. 301-392. Regulation for biologicmaterial, comprising products made from the tissues of animals isprovided under 42 U.S.C 262. Similar approval is required by mostforeign countries. Regulations vary from country to country, but theindividual procedures are well known to those in the art.

[0137] Dosage and Route of Administration

[0138] The dosage to be administered depends to a large extent on thecondition and size of the subject being treated as well as the frequencyof treatment and the route of administration. Regimens for continuingtherapy, including dose and frequency may be guided by the initialresponse and clinical judgment. The parenteral route of injection intothe interstitial space of tissues is preferred, although otherparenteral routes, such as inhalation of an aerosol formulation, may berequired in specific administration, as for example to the mucousmembranes of the nose, throat, bronchial tissues or lungs.

[0139] In preferred protocols, a formulation comprising the nakedpolynucleotide in an aqueous carrier is injected into tissue in amountsof from 10 μl per site to about 1 ml per site. The concentration ofpolynucleotide in the formulation is from about 0.1 μg/ml to about 20mg/ml.

[0140] Regulation of TGT

[0141] Just as DNA based gene transfer protocols require appropriatesignals for transcribing (promoters, enhancers) and processing (splicingsignals, polyadenylation signals) the mRNA transcript, mRNA based TGTrequires the appropriate structural and sequence elements for efficientand correct translation, together with those elements which will enhancethe stability of the transfected mRNA.

[0142] In general, translational efficiency has been found to beregulated by specific sequence elements in the 5′ non-coding oruntranslated region (5′UTR) of the RNA. Positive sequence motifs includethe translational initiation consensus sequence (GCC)^(A)CCATGG (Kozak,Nucleic Acids Res.15:8125 (1987)) and the 5^(G) 7 methyl GpppG capstructure (Drummond et al., Nucleic Acids Res. 13:7375 (1985)). Negativeelements include stable intramolecular 5′ UTR stem-loop structures(Muesing et al., Cell 48:691(1987)) and AUG sequences or short openreading frames preceded by an appropriate AUG in the 5′ UTR (Kozak,Supra, Rao et al., Mol. and Cell. Biol. 8:284(1988)). In addition,certain sequence motifs such as the beta globin 5′ UTR may act toenhance translation (when placed adjacent to a heterologous 5′ UTR) byan unknown mechanism. There are also examples of specific 5′ UTRsequences which regulate eukaryotic translational efficiency in responseto environmental signals. These include the human ferritin 5′ UTR(Hentze et al., Proc. Natl. Acad. Sci. USA 84:6730 (1987)) and thedrosophila hsp⁷0 5′ UTR (Klemenz et al., EMBO Journal 4:2053 (1985)).Finally, there are viral 5′ UTR sequences which are able to bypassnormal cap dependant translation and translational controls and mediateann efficient translation of viral or chimeric mRNAs (Dolph et al., J.of Virol. 62:2059 (1988)), Pelletier and Sonnenberg, Nature 334, 320(1988)). mRNA based TGT protocols must therefore include appropriate 5′UTR translational elements flanking the coding sequence for the proteinof interest.

[0143] In addition to translational concerns, mRNA stability must beconsidered during the development of mRNA based TGT protocols. As ageneral statement, capping and 3′ polyadenylation are the major positivedeterminants of eukaryotic mRNA stability (Drummond, supra; Ross, Mol.Biol. Med. 5:1(1988)) and function to protect the 5′ and 3′ ends of themRNA from degradation. However, regulatory elements which affect thestability of eukaryotic mRNAs have also been defined, and therefore mustbe considered in the development of mRNA TGT protocols. The most notableand clearly defined of these are the uridine rich 3′ untranslated region(3′ UTR) destabilizer sequences found in many short half-life mRNAs(Shaw and Kamen Cell 46:659 (1986)), although there is evidence thatthese are not the only sequence motifs which result in mRNAdestabilization (Kabnick and Housman, Mol. and Cell. Biol. 8:3244(1988)). In addition, specific regulatory sequences which modulatecellular mRNA half life in response to environmental stimuli have alsobeen demonstrated. These include the estrogen mediated modulation ofVitellogenin mRNA stability (Brock and Shapiro, Cell 34:207 (1983)), theiron dependant regulation of transferrin receptor mRNA stability(Mullner and Kuhn, Cell 53:815 (1988)) which is due to a specific 3′ UTRmotif, the prolactin mediated control of Casein mRNA stability (Guyetteet al., Cell 17:1013 (1989)), the regulation of Fibronectin mRNAstability in response to a number of stimuli (Dean et al., J. Cell.Biol. 106:2159 (1988)), and the control of Histone mRNA stability(Graves et al., Cell 48:615 (1987)). Finally, just as viral RNAsequences have evolved which bypass normal eukaryotic mRNA translationalcontrols, likewise some viral RNA sequences seem to be able to conferstability in the absence of 3′ polyadenylation (McGrae and Woodland,Eur. J. of Biochem. 116: 467 (1981)). Some 5′, such as EMC, according toExample 21, are known to function without a cap. This cacophony ofstability modulating elements must also be carefully considered indeveloping mRNA based TGT protocols, and can be used to modulate theeffect of an mRNA treatment.

[0144] Liposome-forming Materials

[0145] The science of forming liposomes is now well developed. Liposomesare unilamellar or multilamellar vesicles, having a membrane portionformed of lipophilic material and an interior aqueous portion. Theaqueous portion is used in the present invention to contain thepolynucleotide material to be delivered to the target cell.

[0146] It is preferred that the liposome forming materials used hereinhave a cationic group, such as a quaternary ammonium group, and one ormore lipophilic groups, such as saturated or unsaturated alkyl groupshaving from about 6 to about 30 carbon atoms. One group of suitablematerials is described in European Patent Publication No. 0187702. Thesematerials have the formula:

[0147] wherein R¹ and R² are the same or different and are alkyl oralkenyl of 6 to 22 carbon atoms, R³, R⁴, and R⁵ are the same ordifferent and are hydrogen, alkyl of 1 to 8 carbons, aryl, aralkyl of 7to 11 carbons, or when two or three of R³, R⁴, and R⁵ are taken togetherthey form quinuclidino, piperidino, pyrrolidino, or morpholino; n is 1to 8, and X is a pharmaceutically acceptable anion, such as a halogen.These compounds may be prepared as detailed in the above-identifiedpatent application; alternatively, at least one of these compounds,N-(2,3-di-(9-(Z)-octadecenyloxy))-prop-1-yl-N,N,N-trimethylammoniumchloride (DOTMA), is commercially available from Bethesda ResearchLaboratories (BRL), Gaithersburg, Md. 20877, USA.

[0148] These quaternary ammonium diether compounds, however, do havesome drawbacks. Because of the ether linkages, they are not readilymetabolized in vivo. When long-term therapy is contemplated, there issome possibility that these materials could accumulate in tissue,ultimately resulting in lipid storage disease and toxic side effects.Accordingly, a preferred class of compositions for use in the presentinvention has the formula:

[0149] wherein R¹ and R² are the same or different and are alkyl oralkenyl of 5 to 21 carbon atoms, R³, R⁴, and R⁵ are the same ordifferent and are hydrogen, alkyl of 1 to 8 carbons, aryl, aralkyl of 7to 11 carbons, or when two or three of R³, R⁴, and R⁵ are taken togetherthey form quinuclidino, piperidino, pyrrolidino, or morpholino; n is 1to 8, and X is a pharmaceutically acceptable anion, such as a halogen.These compounds may be prepared using conventional techniques, such asnucleophilic substitution involving a carboxylic acid and an alkylhalide, by transesterification, or by condensation of an alcohol with anacid or an acid halide.

[0150] Moreover, many suitable liposome-forming cationic lipid compoundsare described in the literature. See, e.g., L. Stamatatos, et al.,Biochemistry 27:3917-3925 (1988); H. Eibl, et al., Biophysical Chemistry10:261-271 (1979).

[0151] Liposome Preparation

[0152] Suitable liposomes for use in the present invention arecommercially available. DOTMA liposomes, for example, are availableunder the trademark Lipofectin from Bethesda Research Labs,Gaithersburg, Md.

[0153] Alternatively, liposomes can be prepared from readily-availableor freshly synthesized starting materials of the type previouslydescribed. The preparation of DOTAP liposomes is detailed in Example 6.Preparation of DOTMA liposomes is explained in the literature, see,e.g., P. Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413-7417.Similar methods can be used to prepare liposomes from other cationiclipid materials. Moreover, conventional liposome forming materials canbe used to prepare liposomes having negative charge or neutral charge.Such materials include phosphatidyl choline, cholesterol,phosphatidyl-ethanolamine, and the like. These materials can alsoadvantageously be mixed with the DOTAP or DOTMA starting materials inratios from 0% to about 75%.

[0154] Conventional methods can be used to prepare other, noncationicliposomes. These liposomes do not fuse with cell walls as readily ascationic liposomes. However, they are taken up by macrophages in vivo,and are thus particularly effective for delivery of polynucleotide tothese cells. For example, commercially dioleoylphosphatidyl choline(DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15° C. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

[0155] The present invention is described below in detail using the 23examples given below; however, the methods described are broadlyapplicable as described herein and are not intended to be limited by theExamples.

EXAMPLE 1 Preparation of Lioposme-Forming DOTAP

[0156] The cationic liposome-forming material1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP) is prepared asreported by L. Stamatatos, et al. (supra) or H. Eibl, et al. (supra).

[0157] Briefly, Stamatatos, et al. report that 1 mmol of3-bromo-1,2-propanediol (Aldrich) was acylated for 48 hours at 20° C.with 3 mmol of oleyl chloride (freshly prepared from oleic acid andoxaloyl chloride) in dry, alcohol-free diethyl ether (20 ml) containing5 mmol of dry pyridine. The precipitate of pyridinium hydrochloride wasfiltered off, and the filtrate was concentrated under nitrogen andredissolved in 10 ml of hexane. The hexane solution was washed 3 timeswith an equal volume of 1:1 methanol/0.1 N aqueous NCOONa, pH 3.0, 3times with 1:1 methanol/0.1 N aqueous NaOH, an d1 time with 1% aqueousNaCl. The crude 3-bromo-1,2-bis-(oleolyloxy)propane was then stirred for72 hours in a sealed tube with a solution of 15% trimethylamine in drydimethyl sulfoxide (30 ml) at 25° C. The products of this reaction weredissolved in chloroform (200 ml), which was repeatedly washed with 1:1methanol/100 mM aqueous HCOONa, pH 3.0, and then evaporated in vacuo toyield a light yellow oil. This material was purified on a column ofsilicic acid (Bio-Sil A, Bio-Rad Laboratories), eluting with a 0-15%gradient of methanol in chloroform to give the desired product in pureform at 9-10% methanol. The purified product was a colorless, viscousoil that migrates with an R_(f) of 0.4 on thin layer chromatographyplates (silica gel G) that were developed with 50:15:5:5:2CHCl₃/acetone/CH₃OH/CH₃COOH/H₂O.

EXAMPLE 2 Preparation of Plasmids for Making DNA Templates for any Geneof Interest

[0158] Suitable template DNA for production of mRNA coding for a desiredpolypeptide may be prepared in accordance with standard recombinant DNAmethodology. As has been previously reported (P. Kreig, et al., NucleicAcids Res. 12:7057-7070 (1984)), a 5′ cap facilitates translation of themRNA. Moreover, the 3′ flanking regions and the poly A tail are believedto increase the half life of the mRNA in vivo.

[0159] The readily-available SP6 cloning vector pSP64T provides 5′ and3′ flanking regions from β-globin, an efficiently translated mRNA. Theconstruction of this plasmid is detailed by Kreig, et al. (supra), andis hereby incorporated by this reference. Any cDNA containing aninitiation codon can be introduced into this plasmid, and mRNA can beprepared from the resulting template DNA. This particular plasmid can becut with BglII to insert any desired cDNA coding for a polypeptide ofinterest.

[0160] Although good results can be obtained with pSP64T when linearizedand then transcribed in vivo with SP6 RNA polymerase, we prefer to usethe xenopus β-globin flanking sequences of pSP64T with phage T7 RNApolymerase. These flanking sequences are purified from pSP64T as thesmall (approx. 150 bp) HindIII to EcoRI fragment. These sequences arethen inserted into a purified linear HindIII/EcoRI fragment (approx. 2.9k bp) from pIBI 31 (commercially available from InternationalBiotechnologies, Inc., Newhaven, Conn. 06535) with T4 DNA ligase.Resulting plasmids, designated pXBG, are screened for orientation andtransformed into E. coli. These plasmids are adapted to receive any geneof interest at a unique BglII restriction site, which is situatedbetween the two xenopus β-globin sequences.

EXAMPLE 3 Preparation of Plasmid Coding for ChloramphenicolAcetyltransferase

[0161] A convenient marker gene for demonstrating in vivo expression ofexogenous polynucleotides is chloramphenicol acetyltransferase, CAT. Aplasmid pSP-CAT containing the CAT gene flanked by the xenopus β-globin5′ and 3′ sequences was produced by adding the CAT gene into the BgIIIsite of pSP64T. We used CAT gene in the form of the small BamHI/HindIIIfragment from pSV2-CAT (available from the American Type CultureCollection, Rockville, Md., Accession No. 37155). However, the CAT geneis commonly used in molecular biology and is available from numeroussources. Both the CAT BamHI/HindIII fragment and the BgIII-cleavedpSP64T were incubated with the Klenow fragment to generate blunt ends,and were then ligated with T4 DNA ligase to form pSP-CAT.

[0162] The small PstI/HindIII fragment was then generated and purified,which comprises the CAT gene between the 5′ and 3′ β-globin flankingsequences of pSP64T. pIBI31 (International Biotechnologies, Inc.) wascleaved with PstI and HindIII, and the long linear sequence waspurified. This fragment was then combined with the CAT-gene containingsequence and the fragments were ligated with T4 DNA ligase to form aplasmid designated pT7CAT An. Clones are selected on the basis ofβ-galactosidase activity with Xgal and ampicillin resistance.

EXAMPLE 4 Preparation of Purified DNA Template

[0163] The plasmid DNA from Example 3 is grown up and prepared as perManiatis (supra), except without RNAse, using 2 CsCl spins to removebacterial RNA. Specifically, E. coli containing pT7CAT An from Example 3was grown up in ampicillin-containing LB medium. The cells were thenpelleted by spinning at 5000 rpm for 10 min. in a Sorvall RC-5centrifuge (E. I. DuPont, Burbank, Calif. 91510), resuspended in coldTE, pH 8.0, centrifuged again for 10 min. at 5000 rpm., resuspended in asolution of 50 mM glucose, 25 mM Tris-Cl pH 8.0, 10 mM EDTA, and 40mg/ml lysozyme. After incubation for 5 to 10 minutes with occasionalinversion, 0.2 N NaOH containing 1% SDS was added, followed after 10minutes at 0° C. with 3 M potassium acetate and 2 M acetic acid. After10 more minutes, the material was again centrifuged at 6000 rpm, and thesupernatant was removed with a pipet. The pellet was then mixed into 0.6vol. isopropanol (−20° C.), mixed, and stored at −20° C. for 15 minutes.The material was then centrifuged again at 10,000 rpm for 20 min., thistime in an HB4 swinging bucket rotor apparatus (DuPont, supra) afterwhich the supernatant was removed and the pellet was washed in 70% EtOHand dried at room temperature. Next, the pellet was resuspended in 3.5ml TE, followed by addition of 3.4 g CsCl and 350 μl of 5 mg/ml EtBr.The resulting material was placed in a quick seal tube, filled to thetop with mineral oil. The tube was spun for 3.5 hours at 80,000 rpm in aVTi80 centrifuge (Beckman Instruments, Pasadena, Calif., 91051). Theband was removed, and the material was centrifuged again, making up thevolume with 0.95 g CsCl/ml and 0.1 ml or 5 mg/ml EtBr/ml in TE. The EtBrwas then extracted with an equal volume of TE saturated N-Butanol afteradding 3 volumes of TE to the band, discarding the upper phase until theupper phase is clear. Next, 2.5 vol. EtOH was added, and the materialwas precipitated at −20° C. for 2 hours. The resultant DNA precipitateis used as a DNA template for preparation of mRNA in vitro.

EXAMPLE 5 Preparation of mRNA for Transfection

[0164] The DNA from Example 4 was linearized downstream of the poly Atail with a 5-fold excess of PstI. The linearized DNA was then purifiedwith two phenol/chloroform extractions, followed by two chloroformextractions. DNA was then precipitated with NaOAc (0.3 M) and 2 volumesof EtOH. The pellet was resuspended at about 1 mg/ml in DEP-treateddeionized water.

[0165] Next, a transcription buffer was prepared, comprising 400 mM TrisHCl (pH 8.0), 80 MM MgCl₂, 50 mM DTT, and 40 mM spermidine. Then, thefollowing materials were added in order to one volume of DEP-treatedwater at room temperature: 1 volume T7 transcription buffer, preparedabove; rATP, rCTP, and rUTP to 1 mM concentration; rGTP to 0.5 mMconcentration; 7meG(5′)ppp(5′)G cap analog (New England Biolabs,Beverly, Mass., 01951) to 0.5 mM concentration; the linearized DNAtemplate prepared above to 0.5 mg/ml concentration; RNAsin (Promega,Madison, Wis.) to 2000 U/ml concentration; and T7 RNA polymerase (N.E.Biolabs) to 4000 U/ml concentration.

[0166] This mixture was incubated for 1 hour at 37 C. The successfultranscription reaction was indicated by increasing cloudiness of thereaction mixture.

[0167] Following generation of the mRNA, 2 U RQ1 DNAse (Promega) permicrogram of DNA template used was added and was permitted to digest thetemplate for 15 minutes. Then, the RNA was extracted twice withchloroform/phenol and twice with chloroform. The supernatant wasprecipitated with 0.3 M NaOAc in 2 volumes of EtOH, and the pellet wasresuspended in 100 μl DEP-treated deionized water per 500 μltranscription product. This solution was passed over an RNAse-freeSephadex G50 column (Boehringer Mannheim #100 411). The resultant mRNAwas sufficiently pure to be used in transfection of vertebrates in vivo.

EXAMPLE 6 Preparation of Liposomes

[0168] A number of liposome preparation methods can be used to advantagein the practice of the present invention. One particularly preferredliposome is made from DOTAP as follows:

[0169] A solution of 10 mg dioleoyl phosphatidylethanolamine (PE) and 10mg DOTAP (from Example 1) in 1 ml chloroform is evaporated to drynessunder a stream of nitrogen, and residual solvent is removed under vacuumovernight. Liposomes are prepared by resuspending the lipids indeionized water (2 ml) and sonicating to clarity in a closed vial. Thesepreparations are stable for at least 6 months.

[0170] Polynucleotide complexes were prepared by mixing 0.5 mlpolynucleotide solution (e.g., from Example 5) at 0.4 mg/ml by slowaddition through a syringe with constant gentle vortexing to a 0.5 mlsolution of sonicated DOTMA/PE or DOTAP/PE liposomes at 20 mg/ml, atroom temperature.

What is claimed is:
 1. A pharmaceutical product, comprising: nakedpolynucleotide, operatively coding for a biologically activepolypeptide, in physiologically acceptable administrable form, in acontainer; and a notice associated with said container in formprescribed by a governmental agency regulating the manufacture, use, orsale of pharmaceuticals, which notice is reflective of approval by saidagency of said form of said polynucleotide for human or veterinaryadministration.
 2. A pharmaceutical product, comprising: nakedpolynucleotide, operatively coding for a biologically active peptide, insolution in a physiologically acceptable injectable carrier and suitablefor introduction interstitially into a tissue to cause cells of saidtissue to express said polypeptide; a container enclosing said solution;and a notice associated with said container in form prescribed by agovernmental agency regulating the manufacture, use, or sale ofpharmaceuticals, which notice is reflective of approval by said agencyof manufacture, use, or sale of said solution of polynucleotide forhuman or veterinary administration.
 3. The product of claim 2, whereinsaid peptide is immunogenic and administration of said solution to ahuman serves to vaccinate said human.
 4. The product of claim 2, whereinsaid peptide is therapeutic and administration of said solution to ahuman in need of therapy relating to said polypeptide has a therapeuticeffect.
 5. A pharmaceutical product for treatment of muscular dystrophy,comprising: a sterile, pharmaceutically acceptable carrier; apharmaceutically effective amount of a naked polynucleotide operativelycoding for dystrophin solubilized in said carrier; and a containerenclosing said carrier and said polynucleotide in sterile fashion. 6.The product of claim 5, wherein said polynucleotide is DNA.
 7. Apharmaceutical product for use in supplying a biologically activepolypeptide to a vertebrate, comprising: a pharmaceutically effectiveamount of a naked polynucleotide operatively coding for saidpolypeptide; a container enclosing said polynucleotide in a sterilefashion; and means associated with said container for permittingtransfer of said polynucleotide from said container to the interstitialspace of a tissue, whereby cells of said tissue can take up and expresssaid polynucleotide.
 8. The product of claim 7, wherein said containeris a syringe.
 9. The product of claim 7, wherein the amount of saidpolynucleotide in said container is at least 5 micrograms.
 10. Apharmaceutical product for use in immunizing a vertebrate, comprising: apharmaceutically effective amount of a naked polynucleotide operativelycoding for an immunogenic polypeptide; a sealed container enclosing saidpolynucleotide in a sterile fashion; and means associated with saidcontainer for permitting transfer of said polynucleotide from saidcontainer to the interstitial space of a tissue, whereby cells of saidtissue can take up and express said polynucleotide.
 11. The product ofclaim 10, wherein said container is a syringe.
 12. The method of claim10, wherein the amount of said polynucleotide in said container is atleast 5 micrograms.
 13. A method for delivering a pharmaceutical orimmunogenic polypeptide to the interior of a cell of a vertebrate invivo, comprising the step of: introducing a preparation comprising apharmaceutically acceptable injectable carrier and a nakedpolynucleotide operatively coding for said polypeptide into theinterstitial space of a tissue comprising said cell, whereby said nakedpolynucleotide is taken up into the interior of said cell and has animmunogenic or pharmacological effect on said vertebrate.
 14. The methodof claim 13, wherein said polypeptide is immunogenic and said vertebratedevelops an immune response to said polypeptide.
 15. The method of claim13, wherein said polypeptide is therapeutic.
 16. The method of claim 13,wherein said polynucleotide is mRNA.
 17. The method of claim 13, whereinsaid polynucleotide is DNA.
 18. The method of claim 13, wherein saidpolynucleotide is a DNA sequence incorporated into a plasmid vector andsaid plasmid vector further comprises a replicator.
 19. The method ofclaim 13, wherein said DNA sequence contains a promoter sequence. 20.The method of claim 19, wherein said promoter is a cell-specificpromoter that permits substantial transcription of DNA only inpredetermined cells.
 21. The method of claim 13, wherein saidpolynucleotide sequence contains a sequence operatively coding for thesecretion of said polypeptide.
 22. The method of claim 13, wherein saidpolypeptide expression is transitory.
 23. The method of claim 13,wherein said polypeptide is an enzyme.
 24. The method of claim 13,wherein said polypeptide is an hormone.
 25. The method of claim 13,wherein said polypeptide is a lymphokine.
 26. The method of claim 13,wherein said polypeptide is a cell surface receptor.
 27. The method ofclaim 13, wherein said polypeptide is a growth factor.
 28. The method ofclaim 13, wherein said polypeptide is a regulatory protein.
 29. Themethod of claim 13, wherein said polynucleotide is incorporated intomuscle cells.
 30. The method of claim 13, wherein said preparation isinjected intramuscularly.
 31. The method of claim 13, wherein saidpolynucleotide is incorporated into cells of skin, brain, lung, liver,spleen or blood.
 32. The method of claim 13, wherein said preparation isinjected intradermally, subdermally, intrathecally, or intravenously.33. The method of claim 13, wherein said preparation is impressed intothe skin.
 34. The method of claim 13, wherein said preparation isdelivered transdermally.
 35. The method of claim 13, wherein saidpolynucleotide is a non-replicating DNA sequence operatively coding forsaid polypeptide and for a polymerase for transcribing said DNA, andwherein said DNA includes recognition sites for said polymerase, andsaid injectable preparation further includes a means for providing aninitial quantity of said polymerase in said cell.
 36. The method ofclaim 35, wherein said polymerase is phage T7 polymerase and saidrecognition site is a T7 origin of replication sequence.
 37. A methodfor treating a disease associated with the deficiency or absence of aspecific polypeptide in a vertebrate, comprising the step of:introducing an injectable preparation comprising a pharmaceuticallyacceptable carrier and containing a naked polynucleotide operativelycoding for said polypeptide into a vertebrate and permitting saidpolynucleotide to be incorporated into a cell, wherein said polypeptideis formed as the translation product of said polynucleotide and saiddeficiency or absence of said polypeptide is effectively treated. 38.The method of claim 37, wherein said preparation is injected intomuscle.
 39. The method of claim 37, wherein said cell is a persistentnon-dividing cell.
 40. A therapy for phenylketonuria according to themethod set forth in claim 37, wherein said polynucleotide codes for thepolypeptide phenylalanine hydroxylase.
 41. The method of claim 37,wherein said polynucleotide codes for human growth hormone.
 42. Atherapy for hypercholesterolemia according to the method set forth inclaim 37, wherein a polynucleotide operatively coding for a receptorinvolved in cholesterol homeostasis is incorporated into a hepatocyte,whereby said receptor is expressed by said cell.
 43. A method forimmunizing a vertebrate, comprising the step of: introducing aninjectable preparation comprising a pharmaceutically acceptable carrierand a naked, expressible polynucleotide operatively coding for animmunogenic peptide interstitially into tissue of a vertebrate wherebyan immunogenic translation product of said polynucleotide is formed by acell of said tissue, thereby eliciting an immune response against saidimmunogen.
 44. The method of claim 43, wherein said immunogenictranslation product is presented by said cell in the context of themajor histocompatibility complex.
 45. The method of claim 43, whereinsaid cells are muscle cells.
 46. The method of claim 43, wherein saidinjectable preparation further comprises an adjuvant or a lymphokine.47. The method of claim 43, wherein said immunogenic translation productis presented by a cell and provokes a humoral immune response,comprising the synthesis of antibody.
 48. The method of claim 43,wherein said cell expresses major histocompatibility antigens of ClassI, and said immunogenic peptide is presented in the context of Class Ihistocompatibility antigens and wherein said immune response is cellularand comprises the production of cytotoxic T-cells.
 49. A method forintroducing a polynucleotide into muscle cells in vivo, comprising thesteps of: providing a composition comprising a naked polynucleotide in apharmaceutically acceptable carrier; and contacting said compositionwith muscle tissue of a vertebrate in vivo, whereby said polynucleotideis introduced into muscle cells of said tissue.
 50. The method of claim49, wherein said polynucleotide is an antisense polynucleotide.
 51. Themethod of claim 49, wherein said polynucleotide codes for a therapeuticpeptide that is expressed by said muscle cells after said contactingstep to provide therapy to said vertebrate.
 52. The method of claim 49,wherein said polynucleotide codes for an immunogenic peptide that isexpressed by said muscle cells after said contacting step and whichgenerates an immune response, thereby immunizing said vertebrate.
 53. Amethod for obtaining long term administration of a polypeptide to avertebrate, comprising the step of introducing a naked DNA sequenceoperatively coding for said polypeptide interstitially into tissue ofsaid vertebrate, whereby cells of said tissue produce said polypeptidefor at least 3 months.
 54. The method of claim 53, wherein said cellsproducing said polypeptide are nonproliferating cells.
 55. The method ofclaim 54, wherein said cells are muscle cells.
 56. A method forobtaining transitory expression of a polypeptide in a vertebrate,comprising the step of introducing a naked mRNA sequence operativelycoding for said polypeptide interstitially into tissue of saidvertebrate, whereby cells of said tissue produce said polypeptide forless than about 10 days.
 57. The method of claim 56, wherein said tissueis solid tissue.
 58. A method for treatment of muscular dystrophy,comprising the steps of: introducing a therapeutic amount of acomposition comprising a polynucleotide operatively coding fordystrophin in a pharmaceutically acceptable carrier in vivo into muscletissue of an animal suffering from muscular dystrophy, whereby saidpolynucleotide is taken up into cells of said tissue and dystrophin isproduced in vivo.
 59. The method of claim 58, wherein said compositionis introduced by means of injection.
 60. The method of claim 58, whereinsaid polynucleotide is a naked polynucleotide and said composition isintroduced interstitially into said muscle tissue.
 61. A pharmaceuticalproduct, comprising: naked antisense polynucleotide in physiologicallyacceptable administrable form, in a container; and a notice associatedwith said container in form prescribed by a governmental agencyregulating the manufacture, use, or sale of pharmaceuticals, whichnotice is reflective of approval by said agency of said form of saidpolynucleotide for human or veterinary administration.
 62. Use of nakedpolynucleotide operatively coding for a physiologically activepolypeptide in the preparation of a pharmaceutical for introductioninterstitially into tissue to cause cells comprising said tissue toproduce said polypeptide.
 63. Use according to claim 62, wherein saidpharmaceutical is for introduction into muscle tissue whereby musclecells produce said polypeptide.
 64. Use according to claim 62, whereinsaid peptide is dystrophin and said pharmaceutical is for treatment ofmuscular dystrophy.
 65. Use of naked antisense polynucleotide in thepreparation of a pharmaceutical for introduction interstitially intotissue of a vertebrate to inhibit translation of polynucleotide in cellsof said vertebrate.